The invention concerns methods and reagent kits for the fluorimetric determination of analytes.
There are numerous methods for determining analytes for example for diagnostic applications. One method is to determine the analyte by means of a redox reaction and a redox indicator. In this case an oxidizing or reducing system acts directly on the redox indicator or via a mediator. The presence of the analyte leads to a reduction or oxidation of the redox indicator which enables a qualitative or quantitative determination to be carried out.
Depending on the type of redox indicator that is used, the indicator can be determined by a colorimetric, fluorimetric or electrochemical detection method. Examples of colorimetric detection reagents are heteropolyacids (EP-B-0 431 456), tetrazolium compounds (EP-B-0 574 769), nitrosoaromatic compounds (EP-A-0 620 283), RIND compounds (EP-B-0 190 740), phenazines (WO 93/06487) and indanthrones (EP-B-0 831 327). Examples of electrochemical detection reagents are nitrosoaromatics, phenazines, potassium hexacyanoferrate and benzoquinones (cf. e.g. EP-A-0 441 222 and EP-A-0 505 494). Examples of fluorimetric detection reagents are e.g. resazurin (U.S. Pat. No. 5,912,139), transition metal complexes (Ryabov et al., JBIC 4 (1999) 175-182; Woltman et al., Anal. Chem. 71 (1999) 1504-1512) and scopoletin, esculetin, p-hydroxyphenylacetic acid, di-chlorofluorescein, N-acetyl-3,7-dihydroxyphenoxazine and MNBDH which are used exclusively for the detection of H2O2 (see also R. Haughland, Handbook of Fluorescent Probes and Research Chemicals, 6th edition 1996).
However, the fluorimetric detection reagents known from the prior art have some disadvantages. Thus most known fluorescent indicators require that metabolites such as glucose are determined by detecting H2O2 generated by glucose oxidase. This reaction usually has to be catalytically supported by the enzyme peroxidase and is very prone to interference by electron donors such as urea or bilirubin. The reagents are also not stable for long time periods.
In contrast, redox indicators that allow an oxygen-independent detection of glucose i.e. which directly accept an electron from an oxidizing enzyme instead of oxygen, are advantageous. However, only resazurin and Os and Ru complexes are known to be suitable electron acceptors for this. However, in the case of resazurin the emission bands of the resorufin formed by the redox reaction strongly overlap the absorption bands of non-reacted resazurin which considerably reduces the sensitivity of the analyte determination. The high redox potential of transition metal complexes (e.g. Ru complexes) results in a strong interference by compounds such as ascorbic acid. Their fluorescence efficiency also varies with the oxygen content of the sample.
Furthermore in the case of the previously known fluorescent indicators the excitation light sources used are mainly limited to the UV and green range of light. Thus for example an inadequate number of compounds are known which allow use of the particularly strong blue and red LEDs.